Clinically approved lung surfactant therapies vary widely in composition, from 40 - 80 percent dipalmitoylphosphatidylcholine (DPPC), with equally wide variations in unsaturated and anionic lipids. Variations in lipid and protein quantity and composition in surfactant taken by lavage are associated with severity of disease in neonatal and adult respiratory distress syndrome. However, an understanding of the roles of the lipid and protein components of lung surfactants is still lacking, and it is difficult to relate variations in composition to either optimal replacement formulations or to specific physiological states. In this revised proposal, we hypothesize that SP-B, in combination with SP-C, enhances the formation of a surfactant "reservoir" that remains attached to the interface during monolayer compression. This model suggests that synergistic interactions between the SP-B and SP-C proteins and the anionic and unsaturated lipids allows these lipids to be held in the monolayer at the high surface pressures developed during compression (low surface tensions), while facilitating re- spreading of the monolayers on re-expansion. We have shown that SP- B acts to fold PG monolayers into the subphase, apparently by increasing the flexibility and elasticity of the monolayer in a way not fully understood. SP-C, which is a transmembrane protein, may stabilize this reservoir structure by stitching together the folded monolayer structure to form more stable multilayers. To test these hypotheses, we will use the fluorescence, polarized fluorescence, Brewster angle and atomic force microscopy techniques developed under the first proposal to: (A) determine if unsaturated and/or saturated phosphatidylcholines can be retained in the monolayer through synergistic interactions with SP-B and/or SP-C (B) determine the protein features required to enhance the production of the folds by examining native SP-B, full length synthetic SP-B, the 1-25 amino terminal peptide, an uncharged mutant of SP-B, and the KL4 peptide. (C) Compare the results of these observations with the morphological characteristics of calf lung surfactant as a benchmark for "natural" surfactant morphology and function. If large fractions of unsaturated lipids can be retained in the monolayer, it is likely that these lipids are necessary to enhance the spreading properties. To test this hypothesis, we plan to (D) design and build a canal-type monolayer viscometer to quantify bilayer fluidity and respreading. A systematic investigation of monolayer viscosity as a function of protein and unsaturated lipid concentration will provide a good measure of the optimal formulation for rapid respreading and could help explain the large unsaturated lipid fraction in natural surfactant.